Part:BBa_K562009:Experience

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••••• iGEM Dundee 2011

This part was seen work in practice. Synthesis of the seven encoded polypeptides in very small scale cultures (1 ml) was observed by 35-S-Methionine labelling (Figure 1) and the proteins could also be purified from large scale cultures (5 litre) and visualised by Coomassie-style staining of SDS-PAGE gels (Figure 2). GFP was detected inside the purified complex of microcompartment proteins be Western immunoblotting (Figure 3).

Results

E. coli was transformed with BBa_K562009 expressed from pT7.5, and the plasmid pGP1-2 encoding T7 polymerase, and grown aerobically for 3 hours in LB medium. T7 polymerase synthesis was induced by heat shock before all native transcription in E. coli was halted by the addition of rifampicin. 10 minutes later 1 ml of cells was then collected and spiked with 35-S-labelled Methionine. Following a 10 minute incubation the cell pellet was harvested and resuspended in Laemmli disaggregation buffer. Protein labelling was analysed by SDS-PAGE and autoradiography.

This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid.

Figure 1: Transcription and translation of gene products produced from BBa_K562009. Radiolabelled revealed bands of the corresponding sizes to the PduA, PduJ, PduK, PduN, PduT and PduU proteins. PduB can be expressed as a smaller PduB' variant from an alternative translation start site. The RED ARROW points to the lane producing polypeptides from BBa_K562009.

E. coli was transformed with BBa_K562009 and BBa_K562012 and grown anaerobically for 16 hours in LB medium containing 0.4% (w/v) glucose. Cells were harvested, washed and broken in 50 mls B-PER solution before being loaded onto an IMAC column. Bound proteins were eluted with an imidazole gradient and analysed by SDS-PAGE.

Fractions contained all the components of the synthetic microcompartment. The bands shown were positively identified by tryptic peptide mass fingerprinting. In addition, the co-expressed GFP was also identified around 26 kDa (Figure 3). Note that the banding pattern changed depending on whether the sample was boiled. In unboiled samples the PduB protein (which is produced as full-lenth PduB and truncated PduB') remains in an oligomeric state and appears larger than expected on the gel.

Figure 2: Isolation of a synthetic bacterial microcompartment by metal affinity chromatography. Each of PduB, PduU, PduN, and PduK carry hexa-histidine tags. PduA, PduT, and PduJ are not tagged.

Figure 3: Pdu20-GFP co-purifies with the His-tagged microcompartment. Western immunoblotting using a mouse monoclonal antobody raised against GFP clearly identified GFP in the 'purified sample' lane. In this case 'purifed sample' is identical to that loaded in the 'boiled' lane of Figure 2 above. This indicates that PduD20-GFP co-purifies with the microcompartment, most likely because it is targeted inside it by the Pdu20 signal sequence.

In an attempt to visualise the size and shape of the isolated compartments negative stain EM was attempted (Figure 4). Although the sample was rather old (some aggregation was obvious) there were some large regular shapes, as well as squashed complexes, visible. Note that this technique gives a 2D projection and that we do not know how rigid the microcompartments are.

Figure 4: Visualisation of the isolated synthetic bacterial microcompartment by negative stain EM. Protein was adhered to a carbon grid and stained with molybdate.

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